Packages¶
In [1]:
import matplotlib_inline.backend_inline as backend_inline
import IPython.display as disp
# Make set_matplotlib_formats available where Scanpy expects it
disp.set_matplotlib_formats = backend_inline.set_matplotlib_formats
import numpy as np # scientific computing toolkit
import pandas as pd # data analysis toolkit
import scanpy as sc # scanpy is referred to with sc.***
import matplotlib.pyplot as plt # Matplotlib is referred to with plt.***
from scipy import stats # for linear regressions
import seaborn as sns # for easy heatmaps
import scirpy as ir # TCR analysis
import scvelo as scv
#import mudata as md # New data structure
#from mudata import MuData
sc.settings.verbosity = 3 # verbosity: errors (0), warnings (1), info (2), hints (3)
sc.logging.print_header() # check if all needed versions are installed and up to date (old: print_Versions(), changed in new version of scanpy)
results_directory = './Analysis_output/'
results_file = results_directory + '/results_file/write/results.h5ad' # the file that will store the analysis results
| Package | Version |
|---|---|
| matplotlib-inline | 0.1.7 |
| numpy | 2.1.3 |
| pandas | 2.2.3 |
| scanpy | 1.11.0 |
| matplotlib | 3.10.1 |
| scipy | 1.15.2 |
| seaborn | 0.13.2 |
| scirpy | 0.12.2 |
| scvelo | 0.3.3 |
| Dependency | Version |
|---|---|
| leidenalg | 0.10.2 |
| charset-normalizer | 3.4.1 |
| prompt_toolkit | 3.0.50 |
| six | 1.17.0 |
| ipython | 9.0.2 |
| pillow | 11.1.0 |
| yamlordereddictloader | 0.4.2 |
| natsort | 8.4.0 |
| setuptools | 75.8.2 |
| python-dateutil | 2.9.0.post0 |
| adjustText | 1.3.0 |
| tqdm | 4.67.1 |
| parasail | 1.3.4 |
| pytz | 2025.1 |
| rich | 13.9.4 |
| zipp | 3.21.0 |
| packaging | 24.2 |
| decorator | 5.2.1 |
| texttable | 1.7.0 |
| defusedxml | 0.7.1 |
| cycler | 0.12.1 |
| executing | 2.2.0 |
| scikit-learn | 1.5.2 |
| stack-data | 0.6.3 |
| session-info2 | 0.1.2 |
| Pygments | 2.19.1 |
| pycairo | 1.27.0 |
| awkward_cpp | 44 |
| airr | 1.5.1 |
| asttokens | 3.0.0 |
| numba | 0.61.0 |
| statsmodels | 0.14.4 |
| llvmlite | 0.44.0 |
| wcwidth | 0.2.13 |
| joblib | 1.4.2 |
| parso | 0.8.4 |
| igraph | 0.11.8 |
| jedi | 0.19.2 |
| pyparsing | 3.2.1 |
| more-itertools | 10.3.0 |
| pyzmq | 26.2.1 |
| jupyter_core | 5.7.2 |
| h5py | 3.13.0 |
| patsy | 1.0.1 |
| torch | 2.6.0 (2.6.0+cpu) |
| jaraco.text | 3.12.1 |
| jaraco.context | 5.3.0 |
| awkward | 2.7.4 |
| kiwisolver | 1.4.8 |
| attrs | 25.1.0 |
| colorama | 0.4.6 |
| debugpy | 1.8.12 |
| PyYAML | 6.0.2 |
| jaraco.collections | 5.1.0 |
| comm | 0.2.2 |
| psutil | 7.0.0 |
| importlib_metadata | 8.6.1 |
| ipywidgets | 8.1.5 |
| jaraco.functools | 4.0.1 |
| jupyter_client | 8.6.3 |
| networkx | 3.4.2 |
| fsspec | 2025.2.0 |
| pywin32 | 308 |
| Cython | 3.0.12 |
| threadpoolctl | 3.5.0 |
| tornado | 6.4.2 |
| typing_extensions | 4.12.2 |
| platformdirs | 4.3.6 |
| anndata | 0.11.3 |
| traitlets | 5.14.3 |
| Levenshtein | 0.27.1 |
| legacy-api-wrap | 1.4.1 |
| setuptools-scm | 8.2.0 |
| ipykernel | 6.29.5 |
| RapidFuzz | 3.12.2 |
| pure_eval | 0.2.3 |
| Component | Info | | --------- | --------------------------------------------------------------------------------------------- | | Python | 3.11.11 | packaged by conda-forge | (main, Dec 5 2024, 14:06:23) [MSC v.1942 64 bit (AMD64)] | | OS | Windows-10-10.0.26100-SP0 | | CPU | 16 logical CPU cores, AMD64 Family 25 Model 116 Stepping 1, AuthenticAMD | | GPU | No GPU found | | Updated | 2025-04-03 09:10 |
Directory variables¶
In [2]:
import glob
import os
from pathlib import Path
sample_directory = './GEX_VDJ/'
analysis_info = './analysis_info/'
adata_directory = './analysis_output/IMMUN_COM/annotated_files/'
In [3]:
plt.rcParams['figure.figsize']=(6,6) #rescale figures
sc.set_figure_params(dpi=100, dpi_save=400, color_map='viridis', vector_friendly=True, format='eps', ipython_format = 'png2x')
### you can set a default figure directory here for saving output
sc.settings.figdir = "./analysis_output/IMMUN_COM/"
## Figure directory for matplotlib figures/axes
save_figure_sc = "./analysis_output/IMMUN_COM/"
#Use
#plt.savefig(save_figure + 'image.pdf')
save_figure_ir = "./analysis_output/IMMUN_COM/"
#csv directory
read_csv = './analysis_output/IMMUN_COM/'
save_csv = r'./analysis_output/IMMUN_COM/'
Load data¶
In [4]:
adata = sc.read(adata_directory + 'adata_dpmult_batch_updated.h5ad')
Clustering¶
In [5]:
from sklearn.metrics import silhouette_score
def titrate_leiden_resolution(adata):
res_array=list(np.round(np.linspace(.2, 1, 10),1))
silhoutte_list=list()
sc.settings.verbosity = 1
for i,res in enumerate(res_array):
sc.tl.leiden(adata, resolution=res)
if adata.obs.leiden.unique().shape[0]>2:
silhoutte_list.append(silhouette_score(adata.obsm['X_umap'],
adata.obs[f'leiden'],
metric='euclidean'))
else:
silhoutte_list.append(np.nan)
print(str(np.round(((i+1)/len(res_array))*100,1))+"%", end="\r", flush=True)
sns.lineplot(x=res_array, y=silhoutte_list, color='black', markers='o')
In [6]:
titrate_leiden_resolution(adata)
100.0%
In [7]:
sc.tl.leiden(adata, resolution = 0.4)
sc.pl.umap(adata, color='leiden', s=10)
Get genes and clusters from Abdullah et al
In [8]:
gene_df = pd.read_csv(read_csv + 'adbullah_seurat.csv')
gene_df = gene_df[gene_df.avg_log2FC>0]
gene_sets = gene_df.groupby('cluster')['Gene name'].apply(list).to_dict()
In [9]:
# For each cluster signature, compute a score and store it in adata.obs
for cluster, gene_list in gene_sets.items():
print(len(gene_list))
score_name = f'score_cluster_{cluster}'
sc.tl.score_genes(adata, gene_list[:15], score_name=score_name)
37 WARNING: genes are not in var_names and ignored: Index(['Cd27', 'Ms4a6b', 'Gm8369', 'BE692007'], dtype='object') 89 WARNING: genes are not in var_names and ignored: Index(['Klrg1', 'Il18rap', 'Ccl5', 'Jak1', 'Prf1', 'Rap1b', 'Klf2', 'Borcs7'], dtype='object') 19 WARNING: genes are not in var_names and ignored: Index(['S100a4', 'S100a11', 'S100a10', 'Anxa5', 'Lsp1'], dtype='object') 461 WARNING: genes are not in var_names and ignored: Index(['Pttg1'], dtype='object') 689 WARNING: genes are not in var_names and ignored: Index(['Hist1h1b', 'Hist1h2ap', 'Hist1h2ae', 'Hist1h2ab', 'Hist1h1e'], dtype='object') 371 WARNING: genes are not in var_names and ignored: Index(['Satb1', 'Lef1', 'Rps20', 'Ramp1', 'Rpl12', 'Rpl36a', 'Rps29', 'Ccr9'], dtype='object') 167 WARNING: genes are not in var_names and ignored: Index(['Trim30a', 'Samhd1', 'Phf11b'], dtype='object')
In [10]:
sns_cmap = sns.color_palette('light:mediumblue', as_cmap = True)
#sns_cmap = sns.color_palette('light:slateblue', as_cmap = True)
#sns_cmap = sns.color_palette('light:darkslateblue', as_cmap = True)
In [11]:
# Create a list of score column names
score_cols = [f'score_cluster_{cluster}' for cluster in gene_sets.keys()]
sc.pl.umap(adata, color=score_cols + ['leiden'], cmap = sns_cmap, vmin = 0, vmax = [0.6, 1, 1.4, 1.2, 1, 1.2, 0.8], frameon=False)
In [12]:
score_cols = [f'score_cluster_{cluster}' for cluster in gene_sets.keys()]
sns_cmap = sns.color_palette('light:mediumblue', as_cmap = True)
v_max_list = [0.6, 1, 2, 1.2, 1, 1.2, 0.8]
ncols = len(score_cols)
fig, ax = plt.subplots(figsize = (4*ncols,4), ncols = ncols)
for i, score in enumerate(score_cols):
sc.pl.umap(adata, color=score, cmap = sns_cmap, vmin = 0, vmax = v_max_list[i], frameon=False,
show = False, ax=ax[i])
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_all_score_umaps.eps')
Phenotype UMAPs¶
In [13]:
phenotype_list = ['Zeb2','Cx3cr1','Cxcr3','Il7r','Tcf7']
In [14]:
ncols = len(phenotype_list)
fig, ax = plt.subplots(figsize = (4*ncols,4), ncols = ncols)
sns_cmap = sns.color_palette('light:firebrick', as_cmap = True)
v_max_list = [2.2, 2.2, 2.2, 2.2, 2.2]
for i, gene in enumerate(phenotype_list):
sc.pl.umap(adata, color = gene, cmap = sns_cmap, vmin=0, vmax=v_max_list[i], s = 15, frameon=False, show=False, ax=ax[i])
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_all_phenotype_genes_umaps.eps')
Selected UMAPS¶
In [15]:
blue_cmap = sns.color_palette('light:mediumblue', as_cmap = True)
red_cmap = sns.color_palette('light:firebrick', as_cmap = True)
plot_list = ['score_cluster_0-MP','Tcf7','score_cluster_1-TE','Zeb2']
plot_color = [blue_cmap,blue_cmap,red_cmap,red_cmap]
v_max_list = [0.6,2.0,1.0,2.0]
ncols = 4
fig, ax = plt.subplots(figsize = (5*ncols,4), ncols = ncols)
for i, gex in enumerate(plot_list):
sc.pl.umap(adata, color = gex, cmap = plot_color[i], vmin=0, vmax=v_max_list[i], s = 15, frameon=False, show=False, ax=ax[i])
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_selected_umaps.eps')
In [16]:
score_cols = [f'score_cluster_{cluster}' for cluster in gene_sets.keys()]
fig, ax = plt.subplots(ncols = len(score_cols), figsize = (20,4))
for i, score in enumerate(score_cols):
sc.pl.violin(
adata,
keys=score,
groupby='leiden',
jitter=True, # add some jitter for better visualization of individual points
stripplot=True, # optionally overlay a strip plot of the data points
show=False,
ax=ax[i]
)
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_score_dis_dpmult.eps')
HVGs
In [17]:
sc.tl.dendrogram(adata, groupby='leiden')
sc.tl.rank_genes_groups(adata, 'leiden', method='wilcoxon', layer = 'log1p_norm')
In [18]:
fig, ax = plt.subplots(figsize = (12,3.8))
sc.pl.rank_genes_groups_dotplot(adata, n_genes=6, show=False,
ax = ax)
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_deg_groups_dpmult.eps')
Annotate leiden/phenotype¶
In [19]:
# Define your mapping from old cluster labels to new names.
new_labels = {
'0': '0_TE',
'1': '1_EE_like',
'2': '2_MP',
'3': '3_CE',
'4': '4_IE_like',
'5': '5_na1',
'6': '6_na2'
}
color_palette = ["#4477AA", "#66CCEE", "#228833", "#CCBB44", "#EE6677", "#AA3377", "#BBBBBB"]
# Rename the categories in place.
adata.obs['leiden'] = adata.obs['leiden'].cat.rename_categories(new_labels)
Color dict for leiden
In [20]:
leiden_dict = {
'2_MP': "#4477AA", # dark blue
'3_CE': "#66CCEE", # light blue
'0_TE': "#EE6677", # pink/red
'4_IE_like': "#228833", # green
'1_EE_like': "#CCBB44", # yellow
'5_na1': "#eeeeee", # gray
'6_na2': "#eeeeee" # gray
}
clusters = sorted(adata.obs['leiden'].unique(), key=lambda x: int(x.split('_')[0]))
adata.uns['leiden_colors'] = [leiden_dict[cl] for cl in clusters]
In [21]:
fig, ax = plt.subplots(figsize = (6,4))
#color_palette = ["#4477AA", "#66CCEE", "#228833", "#CCBB44", "#EE6677", "#AA3377", "#BBBBBB"]
sc.pl.umap(adata, color='leiden', s=10, show = False, ax=ax, frameon = False)
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_selected_leiden_dp_mult.eps')
Marker genes¶
In [22]:
marker_dict = {
'effector': ['Cx3cr1', 'Klre1', 'Klrg1', 'Zeb2', 'Prf1', 'Gzma', 'Gzmb', 'Lgals1'],
'proliferation': ['Top2a', 'Mki67', 'Stmn1', 'Cdk1', 'Birc5'],
'cytoskeleton_ecm': ['Anax2', 'S100a4', 'Vim', 'Lgals3'],
'ifn_responsive': ['Oas3', 'Irf7', 'Stat1', 'Bst2', 'Ifit1', 'Isg15'],
'memory': ['Slamf6', 'Il7r', 'Cxcr3', 'Ltb', 'Gpr183'],
'naive_survival': ['Tcf7', 'Bcl2', 'Lef1', 'Ccr7', 'Sell']
}
In [23]:
marker_dict = {
'effector': ['Cx3cr1', 'Klre1', 'Zeb2', 'Gzma', 'Gzmb', 'Lgals1'],
'proliferation': ['Top2a', 'Mki67', 'Stmn1', 'Cdk1', 'Birc5'],
'cytoskeleton_ecm': ['Vim', 'Lgals3'],
'ifn_responsive': ['Oas3', 'Irf7', 'Stat1', 'Bst2', 'Ifit1', 'Isg15'],
'memory': ['Slamf6', 'Il7r', 'Cxcr3', 'Ltb', 'Gpr183'],
'naive_survival': ['Tcf7', 'Bcl2', 'Ccr7', 'Sell']
}
In [24]:
fig, ax = plt.subplots(figsize = (10,4.5))
# Plot the dotplot, grouping cells by their 'leiden' cluster
sc.pl.dotplot(
adata,
marker_dict,
groupby='leiden',
layer='log1p_norm',
standard_scale='var', # scales expression per gene for better comparison
dot_max=0.7,
ax = ax,
show = False,
)
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_marker_dis_dp_mult.eps')
In [25]:
sc.tl.dendrogram(adata, groupby='leiden')
sc.tl.rank_genes_groups(adata, 'leiden', method='wilcoxon', layer = 'log1p_norm')
fig, ax = plt.subplots(figsize = (12,3.8))
sc.pl.rank_genes_groups_dotplot(adata, n_genes=6, show=False,
ax = ax)
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_deg_groups_dpmult.eps')
Analysis¶
In [26]:
adata.obs['phenotype'] = adata.obs['leiden'].tolist()
new_labels = {
'2_MP': 'MP',
'3_CE': 'CE',
'0_TE': 'TE',
'4_IE_like': 'IE_like',
'1_EE_like': 'EE_like',
'5_na1': 'na1',
'6_na2': 'na2'
}
adata.obs['phenotype'] = adata.obs['phenotype'].astype('category')
adata.obs['phenotype'] = adata.obs['phenotype'].cat.rename_categories(new_labels)
- calculate enrichment score for each clonotype
- (m/n)/(o/N)
- N = total cells in sample (sample stored in adata.obs.mouse) (In Paper sample meant 1 mouse - here it should be calculated per donor then?, ES was only calculated on clone sizes > 10)
- n = total cells of clonotype (stored as adata.obs.clone_id_aa)
- m = total cells of clonotype in cluster X (cluster is stored in adata.obs.phenotype)
- o = total cells in sample in cluster X
Bias is assigned when:
- ES in MP/ES in TE > 2 (for MP bias and vice versa)
Frequency plot
In [27]:
def get_color(adata, obs, value):
color=adata.uns[obs+'_colors'][sorted(adata.obs[obs].unique().tolist()).index(value)]
return(color)
def plot_frequencies(adata, groupby, subgrouping=None, subgroup_order=None, normalize=True, stacked=True, order=None, kind='bar',
bbox_to_anchor=(1,0.5), celltype_cat='celltype', celltypes_to_plot=[], colors=[], return_df=False, ax='None', figsize=(10,5), legend_columns=1):
"""Plot frequencies of an observable in a stacked bar chart.
This function plot a stacked bar chart for an observable e.g. cell types
and can be grouped or subgrouped according to additional parameters
Parameters
----------
adata : Anndata
Anndata object
groupby : str
observable to group the data on (x-axsis)
subgrouping : str, None (default: None)
observable for subgrouping into separate plots
subgroup_order : list, Series, None (default: None)
order of subgroup categories for individual plots
normalize : bool (default: True)
normalize all cells to max. 1
stacked : bool (default: True)
stack the individual bars
order : list, Series
order of group categories (x-axsis)
kind : str (default: 'bar')
kind of plot to be used
bbox_to_anchor : Tuple (default: (1,0.5))
positioning of the legend
celltype_cat : str (default: 'celltype')
observable containing the value information
celltypes_to_plot : list, Series (default: [])
resrtict the number of categories, default: plot all
colors : list, Series (default: [])
colors for the individual celltypes, default from adata.uns
return_df : bool (default: False)
refurn DataFrame with values
ax : typing.Union[matplotlib.axes._axes.Axes, NoneType], optional (default: None)
matplotlib axes object.
figsize : Tuple (default: (10,5))
size of the figure
legend_columns : int (default: 1)
Number of columns in the legend
Returns
-------
If `return_df==True` a pd.DataFrame object of values
the plot as matplotlib.axes.Axes object
"""
remove_ax=False
if (subgroup_order == None)&(subgrouping!=None):
subgroup_order=adata.obs[subgrouping].unique()
if subgrouping != None:
subgroups=[adata[adata.obs[subgrouping]==x].copy() for x in subgroup_order]
else:
subgroups=[adata.copy()]
subgroup_order=['all cells']
if ax=='None':
fig,ax=plt.subplots(ncols=len(subgroups)+1, figsize=figsize, sharey=True)
remove_ax=True
else:
if len(subgroups)>1:
axx=[]
for i, group in enumerate(subgroups):
axx+=[inset_axes(ax,
height="100%",
width=str(int(1/len(subgroups)*100))+"%", bbox_to_anchor=(i*(1/len(subgroups)),0,0.8,1),
bbox_transform=ax.transAxes)]
ax.remove()
ax=axx
else:
ax=[ax]
final_colors=[]
for data, axes, group in zip(subgroups, ax, subgroup_order):
if celltypes_to_plot==[]:
celltypes_plot=data.obs[celltype_cat].unique().tolist()
else:
celltypes_plot=celltypes_to_plot.copy()
celltypes_plot.sort()
if colors==[]:
if celltype_cat+'_colors' in data.uns.keys():
final_colors=[get_color(data, celltype_cat, value) for value in celltypes_plot]
single_donor_adatas=[0]*data.obs[groupby].unique().shape[0]
single_donor_adatas_names=data.obs[groupby].unique().tolist()
for i, samplename in enumerate(single_donor_adatas_names):
single_donor_adatas[i]=data[data.obs[groupby]==samplename,:]
df=pd.DataFrame()
for i, adata_s in enumerate (single_donor_adatas):
df_temp=pd.DataFrame(single_donor_adatas[i].obs[celltype_cat].value_counts())
df_temp.columns=[single_donor_adatas_names[i]]
df=df.join(df_temp, how='outer')
if normalize == True:
df=df/df.sum()
if order != None:
df=df[order]
if final_colors==[]:
df.T[celltypes_plot].plot(kind=kind, stacked=stacked, title=group, grid=False, ax=axes, width=0.8)
else:
df.T[celltypes_plot].plot(kind=kind, stacked=stacked, title=group, grid=False, color=final_colors, ax=axes, width=0.8)
axes.legend(loc='center left', bbox_to_anchor=bbox_to_anchor, ncol=legend_columns)
if len(subgroups)>1:
for axes in ax[:-2]:
axes.get_legend().remove()
ax[-1].remove()
elif remove_ax==True:
ax[-1].remove()
if return_df==True:
return df
Order by TE frequency¶
In [28]:
fraction_series = adata.obs.groupby('mouse')['leiden'].value_counts(normalize=True)
ordered_mouse_list = fraction_series.xs('0_TE', level=1).sort_values(ascending=False).index.tolist()
In [29]:
fraction_series
Out[29]:
mouse leiden
m41 2_MP 0.403061
0_TE 0.280612
1_EE_like 0.137755
4_IE_like 0.102041
3_CE 0.066327
...
m120 2_MP 0.176020
3_CE 0.160714
4_IE_like 0.112245
5_na1 0.012755
6_na2 0.000000
Name: proportion, Length: 560, dtype: float64
In [30]:
#color_palette = ["#4477AA", "#66CCEE", "#228833", "#CCBB44", "#EE6677", "#AA3377", "#BBBBBB"]
#sc.pl.umap(adata, color='leiden', s=30, show = False, ax=ax, frameon = False, palette = color_palette)
Annotate mouse labels from m1 to m80:
In [31]:
list(adata.obs.mouse.unique())
Out[31]:
['m52', 'm57', 'm54', 'm48', 'm43', 'm46', 'm53', 'm47', 'm58', 'm44', 'm59', 'm55', 'm41', 'm45', 'm56', 'm42', 'm49', 'm50', 'm60', 'm51', 'm80', 'm76', 'm71', 'm61', 'm75', 'm66', 'm79', 'm67', 'm73', 'm64', 'm78', 'm77', 'm70', 'm63', 'm74', 'm69', 'm62', 'm65', 'm72', 'm68', 'm95', 'm84', 'm90', 'm89', 'm92', 'm99', 'm93', 'm100', 'm96', 'm86', 'm88', 'm83', 'm85', 'm87', 'm82', 'm98', 'm91', 'm81', 'm97', 'm94', 'm117', 'm120', 'm105', 'm108', 'm103', 'm111', 'm102', 'm113', 'm118', 'm107', 'm104', 'm110', 'm119', 'm115', 'm109', 'm101', 'm114', 'm106', 'm112', 'm116']
In [32]:
adata.obs['mouse'] = adata.obs['mouse'].apply(
lambda x: f"m{int(x.lstrip('m')) - 40}"
)
adata.obs['mouse'] = adata.obs['mouse'].astype('category')
In [33]:
list(adata.obs.mouse.unique())
Out[33]:
['m12', 'm17', 'm14', 'm8', 'm3', 'm6', 'm13', 'm7', 'm18', 'm4', 'm19', 'm15', 'm1', 'm5', 'm16', 'm2', 'm9', 'm10', 'm20', 'm11', 'm40', 'm36', 'm31', 'm21', 'm35', 'm26', 'm39', 'm27', 'm33', 'm24', 'm38', 'm37', 'm30', 'm23', 'm34', 'm29', 'm22', 'm25', 'm32', 'm28', 'm55', 'm44', 'm50', 'm49', 'm52', 'm59', 'm53', 'm60', 'm56', 'm46', 'm48', 'm43', 'm45', 'm47', 'm42', 'm58', 'm51', 'm41', 'm57', 'm54', 'm77', 'm80', 'm65', 'm68', 'm63', 'm71', 'm62', 'm73', 'm78', 'm67', 'm64', 'm70', 'm79', 'm75', 'm69', 'm61', 'm74', 'm66', 'm72', 'm76']
In [34]:
fraction_series = adata.obs.groupby('mouse')['leiden'].value_counts(normalize=True)
ordered_mouse_list = fraction_series.xs('0_TE', level=1).sort_values(ascending=False).index.tolist()
In [35]:
fig, ax = plt.subplots(figsize=(25,5))
plot_frequencies(adata, groupby = 'mouse', celltype_cat = 'leiden', ax = ax, order = ordered_mouse_list)
ax.set_ylabel('Fraction')
fig.savefig(save_figure_sc + 'updated_phenotype_dis.eps')
The PostScript backend does not support transparency; partially transparent artists will be rendered opaque.
Clone size normalization¶
In [36]:
#update clone size normalization (add +1 for to avoid clone size < 1)
#size factor normalization: Mean amount of antigen-reactive cells per donor is calculated over the whole dataset
# size factor = (Antigen-reactive size mouse donor)/(mean size over whole dateset)
# normalized clone size = (Measured clone size/size factor) + 1 (shifted by 1, to avoid clone size < 1)
#mean siinfekl reactive size over each donor
mean_size = adata.obs.mouse[adata.obs.specificity=='reactive'].value_counts().mean()
mouse_list = adata.obs.mouse.unique().tolist()
size_factor_list = []
for mouse in mouse_list:
#individual siinfekl reactive repertoire size
donor_size = adata.obs.mouse[np.logical_and(adata.obs.specificity=='reactive',adata.obs.mouse==mouse)].value_counts()[0]
size_factor = donor_size/mean_size
size_factor_list.append(size_factor)
# get size factors
d = {'mouse':mouse_list, 'size_factor':size_factor_list}
df = pd.DataFrame(data=d)
adata.obs['norm_clone_id_aa_size_mouse'] = adata.obs.clone_id_aa_size_mouse
# annotate normalized clone counts
for i, mouse in enumerate(mouse_list):
size_factor = size_factor_list[i]
adata.obs.norm_clone_id_aa_size_mouse[adata.obs.mouse==mouse] = (adata.obs.norm_clone_id_aa_size_mouse[adata.obs.mouse==mouse] / size_factor)
## normalize for lowest clone size to keep identical variance
clone_list = adata.obs.clone_id_aa.unique().tolist()
adata.obs['norm_clone_id_aa_size_mouse_2'] = adata.obs.norm_clone_id_aa_size_mouse
cn_min = adata.obs.norm_clone_id_aa_size_mouse.min()
adata.obs.norm_clone_id_aa_size_mouse_2 = adata.obs.norm_clone_id_aa_size_mouse*(1/cn_min)
Plot counts for each mouse¶
In [37]:
df = pd.DataFrame(adata.obs.mouse.value_counts()).reset_index()
fig, ax = plt.subplots(figsize=(25,2))
sns.barplot(data = df, x = 'mouse', y = 'count', order = ordered_mouse_list, ax=ax)
ax.set_xticklabels(ax.get_xticklabels(), rotation=90);
In [38]:
#fraction_series = adata.obs.groupby('mouse')['leiden'].value_counts(normalize=True)
#ordered_mouse_list = fraction_series.xs('0_TE', level=1).sort_values(ascending=False).index.tolist()
df = pd.DataFrame(adata.obs.mouse.value_counts()).reset_index()
ordered_mouse_list = df.sort_values(by='count', ascending=False).mouse.tolist()
fig, ax = plt.subplots(figsize=(25,3))
sns.barplot(data = df, x = 'mouse', y = 'count', order = ordered_mouse_list, ax=ax, color = '#FFC300')
ax.set_xticklabels(ax.get_xticklabels(), rotation=90, fontsize = 16);
ax.set_ylim(0,980)
ax.grid(False)
ax.set_yticks([0, 200, 400, 600, 800])
ax.set_yticklabels(ax.get_yticklabels(), fontsize = 16);
ax.set_ylabel('Single cell count')
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_selected_cell_counts.eps')
In [39]:
df = pd.DataFrame(adata.obs.groupby('mouse').agg(
n_clones = ('clone_id_aa','nunique'),
)).reset_index()
fig, ax = plt.subplots(figsize=(25,3))
sns.barplot(data = df, x = 'mouse', y = 'n_clones', order = ordered_mouse_list, ax=ax, color = 'silver')
ax.set_xticklabels(ax.get_xticklabels(), rotation=90, fontsize = 16);
#ax.set_ylim(0,50)
ax.grid(False)
#ax.set_yticks([0, 200, 400, 600, 800])
ax.set_yticklabels(ax.get_yticklabels(), fontsize = 16);
ax.set_ylabel('Clone count')
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_selected_clone_counts.eps')
Combine plots and order¶
In [40]:
#fraction_series = adata.obs.groupby('mouse')['leiden'].value_counts(normalize=True)
#ordered_mouse_list = fraction_series.xs('0_TE', level=1).sort_values(ascending=False).index.tolist()
df = pd.DataFrame(adata.obs.mouse.value_counts()).reset_index()
ordered_mouse_list = df.sort_values(by='count', ascending=False).mouse.tolist()
In [41]:
# Create the figure with 2 rows (top: frequencies, bottom: counts)
fig, (ax1, ax2) = plt.subplots(nrows=2, ncols=1, figsize=(25, 7),
sharex=True,
gridspec_kw={'height_ratios': [2, 5]})
df = pd.DataFrame(adata.obs.mouse.value_counts()).reset_index()
sns.barplot(data=df, x='mouse', y='count', order=ordered_mouse_list, ax=ax1, color = 'silver')
ax1.set_ylabel('Cells', fontsize=14)
ax1.axhline(y=200, color = 'navy', linestyle='--', linewidth=2)
plot_frequencies(adata, groupby='mouse', celltype_cat='leiden', ax=ax2, order=ordered_mouse_list)
ax2.set_ylabel('Fraction', fontsize=14)
ax2.set_xlabel('mouse')
ax2.set_xticklabels(ax2.get_xticklabels(), rotation=90, fontsize=12)
ax2.set_title('')
fig.tight_layout()
Combined single cell size¶
In [42]:
#adata_subset = adata[adata.obs.clone_id_aa_size_mouse>2]
df = pd.DataFrame(adata.obs.mouse.value_counts()).reset_index()
fig, ax = plt.subplots(figsize=(5,3), ncols = 2)
sns.boxplot(y='count', data=df, ax = ax[0], palette = ['white'])
sns.stripplot(y='count', data=df, ax = ax[0], palette = ['black'], jitter=True, s = 3)
ax[0].set_xticklabels(['Reactive'])
ax[0].spines['top'].set_visible(False)
ax[0].spines['right'].set_visible(False)
ax[0].set_ylim(0,1000)
ax[0].set_ylabel('T cell clones per mouse')
ax[0].grid(False)
#adata_subset = adata[adata.obs.clone_id_aa_size_mouse>2]
df = pd.DataFrame(adata.obs.mouse.value_counts()).reset_index()
sns.boxplot(y='count', data=df, ax = ax[1], palette = ['white'], fliersize = 0)
sns.stripplot(y='count', data=df, ax = ax[1], palette = ['black'], jitter=True, s = 3)
ax[1].set_xticklabels([''])
ax[1].spines['top'].set_visible(False)
ax[1].spines['right'].set_visible(False)
ax[1].set_yscale('log')
ax[1].set_ylim(10,5000)
ax[1].set_ylabel('Cells per mouse')
ax[1].set_ylabel('T cell clones per mouse')
ax[1].grid(False)
fig.tight_layout()
Calculate enrichment score and annotate in adata¶
In [43]:
# Loop over each donor (stored in adata.obs.mouse)
for donor in adata.obs['mouse'].unique():
# Get boolean index for cells from the current donor
donor_idx = adata.obs['mouse'] == donor
donor_obs = adata.obs.loc[donor_idx]
# Total number of cells for this donor (N)
donor_N = donor_obs.shape[0]
# Clonotype counts for this donor (n for each clonotype)
donor_clone_counts = donor_obs['clone_id_aa'].value_counts().to_dict()
# Phenotype counts for this donor (o for each phenotype)
donor_phenotype_counts = donor_obs['phenotype'].value_counts().to_dict()
# Loop over each phenotype present in this donor's data
for pheno in donor_obs['phenotype'].unique():
# Subset donor cells with the current phenotype
donor_obs_pheno = donor_obs[donor_obs['phenotype'] == pheno]
# Count clonotypes within this phenotype (m for each clonotype)
donor_clone_counts_pheno = donor_obs_pheno['clone_id_aa'].value_counts().to_dict()
# Total cells with the current phenotype (o)
donor_o = donor_phenotype_counts[pheno]
# Dictionary to hold enrichment scores for each clonotype for this phenotype in this donor
enrichment_dict = {}
# Compute enrichment score for each clonotype in the donor
for clone, n in donor_clone_counts.items():
m = donor_clone_counts_pheno.get(clone, 0)
# Avoid division by zero
if n > 0 and donor_o > 0 and m > 0 and donor_N > 0:
enrichment = (m / n) * (donor_N / donor_o)
else:
# if zero division, add +1 to numerator and denominator to allow for score calculation (ratio is kept the same)
enrichment = ((m + 1) / (n + 1)) * ((donor_N + 1) / (donor_o + 1))
enrichment_dict[clone] = enrichment
# Define the new column name (e.g., "Effector_ES" if pheno == "Effector")
col_name = f"{pheno}_ES"
# Initialize the column if it doesn't exist yet (set to NaN)
if col_name not in adata.obs.columns:
adata.obs[col_name] = pd.NA
# Map the enrichment score for each cell in the donor based on its clonotype
adata.obs.loc[donor_idx, col_name] = donor_obs['clone_id_aa'].map(enrichment_dict)
In [44]:
#### check for NaN
# 1. Check if there are *any* NaNs (True/False)
has_nans = adata.obs['MP_ES'].isna().any()
print("Contains NaNs MP ES?", has_nans)
# 2. Count how many NaNs
n_nans = adata.obs['MP_ES'].isna().sum()
print("Number of NaNs MP ES:", n_nans)
has_nans = adata.obs['TE_ES'].isna().any()
print("Contains NaNs TE ES?", has_nans)
n_nans = adata.obs['TE_ES'].isna().sum()
print("Number of NaNs TE ES:", n_nans)
Contains NaNs MP ES? False Number of NaNs MP ES: 0 Contains NaNs TE ES? False Number of NaNs TE ES: 0
In [45]:
#Calculate enrichment ratio
adata.obs['MP_over_TE'] = adata.obs['MP_ES'] / adata.obs['TE_ES']
adata.obs['TE_over_MP'] = adata.obs['TE_ES'] / adata.obs['MP_ES']
In [46]:
#### check for NaN
# 1. Check if there are *any* NaNs (True/False)
has_nans = adata.obs['MP_over_TE'].isna().any()
print("Contains NaNs MP_over_TE?", has_nans)
# 2. Count how many NaNs
n_nans = adata.obs['MP_over_TE'].isna().sum()
print("Number of NaNs MP_over_TE:", n_nans)
has_nans = adata.obs['TE_over_MP'].isna().any()
print("Contains NaNs TE_over_MP?", has_nans)
n_nans = adata.obs['TE_over_MP'].isna().sum()
print("Number of NaNs TE_over_MP:", n_nans)
Contains NaNs MP_over_TE? False Number of NaNs MP_over_TE: 0 Contains NaNs TE_over_MP? False Number of NaNs TE_over_MP: 0
assign enrichment (bias)
In [47]:
# set everything to not_assigned, next filter for criteria to allow for assignment
adata.obs['bias'] = 'not_assigned'
# everything above size 10 is set to unbiased
# when Enrichment and Score ratio is fullfilled assign bias
adata.obs.bias[adata.obs.clone_id_aa_size_mouse>10] = 'unbiased'
adata.obs.bias[np.logical_and(np.logical_and(adata.obs.clone_id_aa_size_mouse>10, adata.obs['MP_over_TE']>2), adata.obs['MP_ES']>1)] = 'mem'
adata.obs.bias[np.logical_and(np.logical_and(adata.obs.clone_id_aa_size_mouse>10, adata.obs['TE_over_MP']>2), adata.obs['TE_ES']>1)] = 'eff'
adata.obs['bias'] = adata.obs['bias'].astype('category')
Export relevant data for enrichment score analysis¶
Calculate sharedness¶
In [48]:
df_shared = adata.obs.groupby('clone_id_aa').agg(
shared_mouse_dp_mult = ('mouse','nunique'),
clonotype = ('clone_id_aa','first'),
)
df_shared = df_shared.dropna()
df_shared = df_shared[df_shared.shared_mouse_dp_mult>1]
df_shared = df_shared.reset_index()
adata.obs['shared_mouse_dp_mult']=1
for clonotype in df_shared.clonotype.tolist():
adata.obs.shared_mouse_dp_mult[adata.obs.clone_id_aa==clonotype] = df_shared.loc[df_shared.clonotype==clonotype, 'shared_mouse_dp_mult'].item()
In [49]:
import venn
dpmult_list = ['1_dp_mult','2_dp_mult','3_dp_mult','4_dp_mult']
c_dict = {}
for i, exp in enumerate(dpmult_list):
c_list = adata.obs.clone_id_aa[adata.obs.experiment==exp].tolist()
c_dict[exp] = set(c_list)
fig, ax = plt.subplots(figsize=(5,6))
venn.venn(c_dict, ax=ax, legend_loc='best')
ax.set_title(str(adata.obs.clone_id_aa.nunique()) + ' identified clones')
print(str(adata.obs.clone_id_aa.nunique()) + ' unique clones')
fig.tight_layout()
2675 unique clones
Repertoire size per mouse
In [50]:
#adata_subset = adata[adata.obs.clone_id_aa_size_mouse>2]
df = pd.DataFrame(adata.obs.groupby('mouse').agg(
n_clones = ('clone_id_aa', 'nunique')
))
fig, ax = plt.subplots(figsize=(1.8,3))
sns.boxplot(y='n_clones', data=df, ax = ax, palette = 'hls')
sns.stripplot(y='n_clones', data=df, ax = ax, palette = ['black'], jitter=True, s = 3)
ax.set_xticklabels(['Reactive'])
ax.spines['top'].set_visible(False)
ax.spines['right'].set_visible(False)
ax.set_ylim(0,70)
ax.set_ylabel('TCR repertoire size per mouse')
fig.tight_layout()
Plot clone size and sharedness in dpmult data¶
Plot only inbetween this data set
In [51]:
import matplotlib as mpl
clone_list = adata.obs.clone_id_aa[np.logical_and(adata.obs.shared_mouse_dp_mult>2, adata.obs.specificity=='reactive')].unique().tolist()
adata_sub = adata[adata.obs.clone_id_aa.isin(clone_list)]
#cmap = sns.dark_palette("#69d", reverse=False, as_cmap=True)
#cmap = sns.dark_palette("#e9af33", reverse=False, as_cmap=True)
n_colors = len(clone_list)
cmap = mpl.cm.get_cmap("Greys_r")
color_palette = [cmap(x) for x in np.linspace(0, 0.6, n_colors)]
df = pd.DataFrame(adata_sub.obs.groupby('clone_id_aa').agg(
clone_id_aa = ('clone_id_aa','first'),
shared_mouse = ('shared_mouse_dp_mult','first'),
)).dropna()
df = df.sort_values(by='shared_mouse', ascending = False)
df = df.reset_index(drop = True)
fig, ax = plt.subplots(figsize = (16,3.2))
order = df.sort_values('shared_mouse', ascending=False)['clone_id_aa']
sns.barplot(data=df, x='clone_id_aa', y='shared_mouse', order=order, ax=ax, palette = color_palette, width = 0.9, fill = True, linewidth=3)
ax.set_xticklabels(ax.get_xticklabels(), rotation=90, fontsize=14.5)
ax.set_yticklabels([0,5,10,15,20,25], fontsize=16)
ax.set_ylim(0,25)
ax.set_ylabel('Shared in n mice')
ax.grid(False)
ax.set_xlabel('')
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_selected_sharedness.eps')
print(len(clone_list))
76
Plot clonal expansion and rank'
In [52]:
df = adata_sub.obs.groupby(['clone_id_aa', 'mouse']).agg(
shared_mouse = ('shared_mouse', 'first'),
clone_id_aa_size_mouse=('clone_id_aa_size_mouse', 'first'),
norm_clone_id_aa_size_mouse=('norm_clone_id_aa_size_mouse_2', 'first'),
clone_id_aa_rank=('clone_id_aa_rank', 'first'),
TE_ES=('TE_ES', 'first'),
MP_ES=('MP_ES', 'first'),
MP_over_TE=('MP_over_TE', 'first'),
TE_over_MP=('TE_over_MP', 'first'),
bias=('bias', 'first'),
).reset_index()
In [53]:
df = df.dropna()
In [54]:
# Convert clone_id_aa to a categorical variable with the specified order
df['clone_id_aa'] = pd.Categorical(df['clone_id_aa'], categories=list(order), ordered=True)
# Plot the dotplot: one dot per entry (using jitter to avoid overlapping)
fig, ax = plt.subplots(figsize = (16,3.2))
sns.stripplot(data=df, x='clone_id_aa', y='norm_clone_id_aa_size_mouse', jitter=True, ax=ax, palette = ['black'])
sns.boxplot(data=df, x='clone_id_aa', y='norm_clone_id_aa_size_mouse', ax=ax, palette = ['gray'], width = 0.3, fill = False, fliersize = 0)
ax.set_xticklabels(ax.get_xticklabels(), rotation=90, fontsize=14.5)
#ax.set_yticklabels(ax.get_yticklabels(), fontsize=16)
ax.set_yscale('log')
ax.set_ylim(0.8, 1000)
ax.set_ylabel('Clone size (norm.)')
ax.grid(False)
ax.set_xlabel('')
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_selected_clone_size.eps')
In [55]:
# Convert clone_id_aa to a categorical variable with the specified order
df['clone_id_aa'] = pd.Categorical(df['clone_id_aa'], categories=list(order), ordered=True)
# Plot the dotplot: one dot per entry (using jitter to avoid overlapping)
fig, ax = plt.subplots(figsize = (16,3.2))
sns.stripplot(data=df, x='clone_id_aa', y='clone_id_aa_size_mouse', jitter=True, ax=ax, palette = color_palette)
ax.set_xticklabels(ax.get_xticklabels(), rotation=90, fontsize=16)
ax.set_yticklabels(ax.get_yticklabels(), fontsize=16)
ax.set_yscale('log')
ax.set_ylim(0.8, 1000)
ax.set_ylabel('Clone size (raw.)')
ax.grid(False)
ax.set_xlabel('')
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_selected_clone_size_raw.eps')
In [56]:
df.to_excel('./IMMUNITY_clone_size_df.xlsx')
In [57]:
df.groupby('clone_id_aa').agg(
mean_norm_size = ('norm_clone_id_aa_size_mouse','mean'),
mean_raw_size = ('clone_id_aa_size_mouse','mean'),
mean_TE_ES = ('TE_ES','mean'),
mean_MP_ES = ('MP_ES','mean'),
).to_excel('./IMMUNITY_clone_size_df_aggregated_mean.xlsx')
Calculate size variance¶
Export data for R analysis and variance calculations
In [58]:
df.norm_clone_id_aa_size_mouse = df.norm_clone_id_aa_size_mouse.astype(float)
In [59]:
relevant_cols = ['clone_id_aa','mouse','shared_mouse','clone_id_aa_size_mouse','norm_clone_id_aa_size_mouse']
df[relevant_cols].to_excel('./updated_data_variance_clone_size.xlsx', index=False)
Get shared clones and mice with bias assignments in at least 3 mice for phenotypic comparison of TCR identical T cell clones between donors¶
In [60]:
bias_list = ['eff', 'mem', 'unbiased']
filtered_clones = adata.obs.groupby('clone_id_aa').filter(
lambda group: group.loc[group['bias'].isin(bias_list), 'mouse'].nunique() >= 3
)
clone_list = filtered_clones.clone_id_aa.unique().tolist()
print(len(clone_list))
22
In [61]:
#subet to clone list
adata_sub = adata[adata.obs.clone_id_aa.isin(clone_list)]
#get consistent clone ordering over plots
df = pd.DataFrame(adata_sub.obs.groupby('clone_id_aa').agg(
clone_id_aa = ('clone_id_aa','first'),
shared_mouse = ('shared_mouse_dp_mult','first'),
)).dropna()
df = df.sort_values(by='shared_mouse', ascending = False)
df = df.reset_index(drop = True)
order = df.sort_values('shared_mouse', ascending=False)['clone_id_aa']
Plot sharedness of new clones
In [62]:
n_colors = len(clone_list)
cmap = mpl.cm.get_cmap("Greys_r")
color_palette = [cmap(x) for x in np.linspace(0, 0.6, n_colors)]
In [63]:
fig, ax = plt.subplots(figsize = (8,4))
sns.barplot(data=df, x='clone_id_aa', y='shared_mouse', order=order, ax=ax, palette = color_palette)
ax.set_xticklabels(ax.get_xticklabels(), rotation=45, ha="right", rotation_mode = 'anchor', fontsize = 16)
ax.axhline(y=3, linewidth=1, color='r', linestyle='--')
ax.set_ylabel('Shared by N mice')
ax.grid(False)
fig.tight_layout()
Get plotting dataframe
In [64]:
df = adata_sub.obs.groupby(['clone_id_aa', 'mouse']).agg(
shared_mouse = ('shared_mouse_dp_mult', 'first'),
clone_id_aa_size_mouse=('clone_id_aa_size_mouse', 'first'),
norm_clone_id_aa_size_mouse=('norm_clone_id_aa_size_mouse_2', 'first'),
clone_id_aa_rank=('clone_id_aa_rank', 'first'),
TE_ES=('TE_ES', 'first'),
MP_ES=('MP_ES', 'first'),
MP_over_TE=('MP_over_TE', 'first'),
TE_over_MP=('TE_over_MP', 'first'),
bias=('bias', 'first'),
).reset_index()
df = df.dropna()
In [65]:
# Convert clone_id_aa to a categorical variable with the specified order
df['clone_id_aa'] = pd.Categorical(df['clone_id_aa'], categories=list(order), ordered=True)
# Plot the dotplot: one dot per entry (using jitter to avoid overlapping)
fig, ax = plt.subplots(figsize = (8,3))
sns.stripplot(data=df, x='clone_id_aa', y='norm_clone_id_aa_size_mouse', jitter=True, ax=ax, palette = color_palette)
ax.set_xticklabels(ax.get_xticklabels(), rotation=45, ha="right", rotation_mode = 'anchor')
ax.set_yscale('log')
ax.set_ylim(0.8, 1000)
ax.set_ylabel('Clone size (norm.)')
# Convert clone_id_aa to a categorical variable with the specified order
df['clone_id_aa'] = pd.Categorical(df['clone_id_aa'], categories=list(order), ordered=True)
# Plot the dotplot: one dot per entry (using jitter to avoid overlapping)
fig, ax = plt.subplots(figsize = (8,3))
sns.stripplot(data=df, x='clone_id_aa', y='clone_id_aa_size_mouse', jitter=True, ax=ax, palette = color_palette)
ax.set_xticklabels(ax.get_xticklabels(), rotation=45, ha="right", rotation_mode = 'anchor')
ax.set_yscale('log')
ax.set_ylim(0.8, 1000)
ax.set_ylabel('Clone size (raw)')
Out[65]:
Text(0, 0.5, 'Clone size (raw)')
Get score distribution over each clone¶
drop not assigned
In [66]:
df = df[df.bias!='not_assigned']
Sort data by sharedness and assigned bias
In [67]:
#df['n_bias'] = df.groupby('clone_id_aa')['bias'].transform('nunique')
df['n_bias'] = df.groupby('clone_id_aa')['bias'].transform('count')
df_order = df.drop_duplicates(subset=['clone_id_aa'])
order = df_order.sort_values(['n_bias','shared_mouse'], ascending=[False,False])['clone_id_aa']
In [68]:
fig, ax = plt.subplots(figsize = (8,4))
sns.barplot(data=df, x='clone_id_aa', y='shared_mouse', order=order, ax=ax, palette = color_palette)
ax.set_xticklabels(ax.get_xticklabels(), rotation=45, ha="right", rotation_mode = 'anchor', fontsize = 16)
ax.axhline(y=3, linewidth=1, color='r', linestyle='--')
ax.set_ylabel('Shared by N mice')
ax.grid(False)
fig.tight_layout()
In [69]:
# Convert clone_id_aa to a categorical variable with the specified order
df['clone_id_aa'] = pd.Categorical(df['clone_id_aa'], categories=list(order), ordered=True)
# Plot the dotplot: one dot per entry (using jitter to avoid overlapping)
fig, ax = plt.subplots(figsize = (8,3.5))
sns.stripplot(data = df, x='clone_id_aa', y='TE_over_MP', jitter=0.2, ax=ax, palette = color_palette, edgecolor = 'black', linewidth = 0, s = 8)
#sns.boxplot(data=df, x='clone_id_aa', y='MP_over_TE', ax=ax, palette = ['gray'], width = 0.3, fill = False, fliersize = 0)
#sns.stripplot(data = df[df.MP_over_TE>2], x='clone_id_aa', y='MP_over_TE', jitter=0.2, ax=ax, palette = color_palette, edgecolor = 'maroon', linewidth = 1.5, s = 8)
#sns.stripplot(data = df[df.MP_over_TE<0.5], x='clone_id_aa', y='MP_over_TE', jitter=0.2, ax=ax, palette = color_palette, edgecolor = 'navy', linewidth = 1.5, s = 8)
ax.axhline(y = 2, linewidth=1, color='maroon', linestyle='--')
ax.axhline(y = 0.5, linewidth=1, color='navy', linestyle='--')
ax.set_xticklabels(ax.get_xticklabels(), rotation=45, ha="right", rotation_mode='anchor', fontsize = 16)
ax.set_yticklabels(ax.get_yticklabels(), fontsize = 16)
ax.set_yscale('log')
ax.set_ylim(0.01, 100)
ax.set_ylabel('TE ES/MP ES')
ax.grid(False)
ax.set_xlabel('')
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_selected_bias_score.eps')
Export for further calculations in R¶
In [70]:
relevant_cols = ['clone_id_aa','mouse','shared_mouse','TE_over_MP','TE_ES','MP_ES']
df[relevant_cols].to_excel('./data_variance_enirchment_score.xlsx',index=False)
In [71]:
leiden_dict = {
'2_MP': "#4477AA", # dark blue
'3_CE': "#66CCEE", # light blue
'0_TE': "#EE6677", # pink/red
'4_IE_like': "#228833", # green
'1_EE_like': "#CCBB44", # yellow
'5_na1': "#EEEEEE", # gray
'6_na2': "#EEEEEE" # gray
}
Frequency of bias per clone¶
In [72]:
color_dict = {
'eff': '#D6604D', # muted red
'mem': '#4575B4', # muted blue
'unbiased': '#D3D3D3', # muted gray
'not_assigned': '#FEE08B' # muted_yellow
}
# Ensure clone_id_aa is a categorical variable with the specified order.
df['clone_id_aa'] = pd.Categorical(df['clone_id_aa'], categories=list(order), ordered=True)
# Create a frequency table for 'bias' for each clone_id_aa.
bias_freq = df.groupby(['clone_id_aa', 'bias']).size().unstack(fill_value=0)
# Reindex the DataFrame to ensure the clone order matches your specified order.
bias_freq = bias_freq.reindex(list(order))
# Normalize each row (each clone) so that the sum equals 100%.
bias_freq = bias_freq.div(bias_freq.sum(axis=1), axis=0) * 100
# Specify the order of the bias categories for plotting
bias_order = ['eff', 'mem', 'unbiased', 'not_assigned']
fig, ax = plt.subplots(figsize=(9, 3))
# Initialize the bottom positions for the stacked bars.
bottom = np.zeros(len(bias_freq))
# Loop over each bias category in the desired order and plot the corresponding segment.
for bias in bias_order:
# Only plot if the bias is present in the DataFrame columns.
if bias in bias_freq.columns:
values = bias_freq[bias].values
ax.bar(bias_freq.index, values, bottom=bottom, color=color_dict[bias], label=bias)
bottom += values
# Formatting the plot.
ax.set_xticklabels(ax.get_xticklabels(), rotation=45, ha="right")
ax.set_ylabel('Frequency (%)')
ax.set_xlabel('clone_id_aa')
ax.legend(title='Bias', bbox_to_anchor=(1.05, 1), loc='upper left')
ax.grid(False)
Frequency of bias per clone but dropped not_assigned
In [73]:
# Create a frequency table for 'bias' for each clone.
bias_freq = df.groupby(['clone_id_aa', 'bias']).size().unstack(fill_value=0)
# Drop the 'not_assigned' column if it exists.
if 'not_assigned' in bias_freq.columns:
bias_freq = bias_freq.drop(columns='not_assigned')
# Reindex the frequency table so that all clones in clone_order appear, even if with all zeros.
bias_freq = bias_freq.reindex(list(order), fill_value=0)
# Define the bias order for plotting (without 'not_assigned').
bias_order = ['eff', 'mem', 'unbiased']
### Plot 1: Normalized Stacked Bar Plot (Percentage Frequency)
# Normalize each clone's counts to 100% (if a clone has all zeros, avoid division by zero by replacing with 1)
bias_freq_norm = bias_freq.div(bias_freq.sum(axis=1).replace(0, 1), axis=0) * 100
fig, ax = plt.subplots(figsize=(9, 3))
bottom = np.zeros(len(bias_freq_norm))
for bias in bias_order:
if bias in bias_freq_norm.columns:
values = bias_freq_norm[bias].values
ax.bar(bias_freq_norm.index, values, bottom=bottom, color=color_dict[bias], label=bias)
bottom += values
ax.set_xticklabels(ax.get_xticklabels(), rotation=45, ha="right", rotation_mode='anchor')
ax.set_ylabel('Frequency (%)')
#ax.set_xlabel('clone_id_aa')
ax.set_title("Normalized Frequency of Bias per Clone")
ax.legend(title='Bias', bbox_to_anchor=(1.05, 1), loc='upper left')
ax.grid(False)
################################################################
##### Show absolute counts
fig, ax = plt.subplots(figsize=(8, 3.5))
bottom = np.zeros(len(bias_freq))
for bias in bias_order:
if bias in bias_freq.columns:
values = bias_freq[bias].values
ax.bar(bias_freq.index, values, bottom=bottom, color=color_dict[bias], label=bias)
bottom += values
ax.set_xticklabels(ax.get_xticklabels(), rotation=45, ha="right", rotation_mode='anchor', fontsize = 16)
ax.set_yticks([0,2,4,6,8,10,12,14])
ax.set_yticklabels(ax.get_yticklabels(), fontsize = 16)
ax.set_ylabel('Phenotype in mouse')
#ax.set_title("Absolute Counts of Bias per Clone")
#ax.legend(title='Bias', bbox_to_anchor=(1.05, 1), loc='upper left')
#ax.set_ylim([0,14])
ax.grid(False)
#ax.get_legend().remove()
fig.tight_layout()
fig.savefig(save_figure_sc + 'updated_selected_bias_category.eps')
Plot UMAPs for these clones¶
Get all clones which appear over these 3 biases (or at least eff and mem) - then filter for clones with high score
In [74]:
bias_list = ['eff', 'mem', 'unbiased']
filtered_clones = pd.DataFrame(adata.obs.groupby('clone_id_aa').filter(
lambda group: set(bias_list).issubset(set(group['bias']))
))
clone_list = filtered_clones.clone_id_aa.unique().tolist()
In [75]:
color_dict = {
'eff': '#D6604D', # muted red
'mem': '#4575B4', # muted blue
'unbiased': 'black', # muted gray
'not_assigned': '#FEE08B' # muted_yellow
}
UMAP KDE Overlay for all shared clones with assigned phenotype bias¶
In [76]:
df = adata_sub.obs.groupby(['clone_id_aa', 'mouse']).agg(
shared_mouse = ('shared_mouse', 'first'),
clone_id_aa_size_mouse=('clone_id_aa_size_mouse', 'first'),
norm_clone_id_aa_size_mouse=('norm_clone_id_aa_size_mouse_2', 'first'),
clone_id_aa_rank=('clone_id_aa_rank', 'first'),
TE_ES=('TE_ES', 'first'),
MP_ES=('MP_ES', 'first'),
MP_over_TE=('MP_over_TE', 'first'),
TE_over_MP=('TE_over_MP', 'first'),
bias=('bias', 'first'),
).reset_index()
df = df.dropna()
df = df[df.bias!='not_assigned'].reset_index(drop=True).copy()
In [77]:
### select only top 3 clones for faster plotting
clone_order = order[:3].tolist()
In [78]:
nrows = len(clone_order)
ncols = len(df[df.clone_id_aa==clone_order[0]].mouse.unique().tolist())
#plotting params
x_all = adata.obsm['X_umap'][:, 0]
y_all = adata.obsm['X_umap'][:, 1]
xmin, xmax = x_all.min(), x_all.max()
ymin, ymax = y_all.min(), y_all.max()
fig, ax = plt.subplots(ncols = ncols, nrows = nrows, figsize = (3 * ncols, 3 * nrows), constrained_layout=True)
#row indexer
y = 0
for clone in clone_order:
#subset dataframe for ordering and scores
mouse_list = df[df.clone_id_aa==clone].mouse.unique().tolist()
#filter for mouse and clone
df_subset = df[np.logical_and(df.mouse.isin(mouse_list), df.clone_id_aa==clone)].copy()
#sort dataframe from highest effector score to unbiased to highest memory score
df_subset['sorted_score'] = df_subset.TE_over_MP.tolist()
df_subset.sorted_score[df_subset.bias == 'mem'] = df_subset[df_subset.bias == 'mem'].MP_over_TE * (-1)
df_subset = df_subset.sort_values(by='sorted_score', ascending = False).copy()
#sorted data
sorted_m = df_subset.mouse.tolist()
sorted_bias = df_subset.bias.tolist()
#start plotting
# col indexer
x = 0
for mouse, bias in zip(sorted_m, sorted_bias):
# Plot background distribution of cells in silver
sc.pl.umap(
adata,
color='leiden',
add_outline=True, outline_width=(0.2, 0), outline_color=('black', 'white'),
s=50,
show=False,
ax=ax[y,x],
frameon=False,
palette=['lightgray'],
legend_loc=None
)
# Extract UMAP coordinates for the subset (clone 'x')
subset = adata[np.logical_and(adata.obs.clone_id_aa == clone, adata.obs.mouse == mouse)]
umap_coords = subset.obsm['X_umap']
if bias == 'eff':
c = 'maroon'
elif bias == 'unbiased':
c = 'black'
else:
c = 'navy'
#get clone size in mouse to adjust threshold
clone_size = df_subset[np.logical_and(df_subset.mouse == mouse, df_subset.bias == bias)].clone_id_aa_size_mouse.values[0]
if clone_size < 20:
kde_thresh = 0.4
levels = 4
else:
kde_thresh = 0.2
levels = 4
#plot density
sns.kdeplot(
x=umap_coords[:, 0],
y=umap_coords[:, 1],
ax=ax[y,x],
fill=True,
color=c,
#cmap=cmap,
bw_adjust = 0.8,
alpha= 0.9, # Adjust transparency if needed
levels = levels,
thresh = kde_thresh,
linewidths = 5,
clip_on = False
)
#adjust colum for new donor and set title
ax[y,x].set_title('M' + mouse[1:], fontsize = 32, loc='center', y=1.0, pad=-0);
x = x+1
#adjust row for new clone
y = y+1
for ax_ in fig.axes:
ax_.set_xlim(-6, 14)
ax_.set_ylim(0, 18)
fig.canvas.draw()
fig.tight_layout(pad=0.4, w_pad=0.5, h_pad=1.0)
for i in range(nrows):
# Get the bounding box of the leftmost axis in this row (in figure coordinates)
bbox = ax[i, 0].get_position()
# Calculate the vertical center of this axis
y_center = (bbox.y0 + bbox.y1) / 2
# Place text slightly to the left of the axis; adjust the x-offset as needed
fig.text(bbox.x0 - 0.02, y_center, clone_order[i],
va='center', ha='right', fontsize=32, rotation='vertical')
fig.savefig(save_figure_sc + 'selected_kdp_umaps_sel_clones_all_clusters.eps')
WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color.
The PostScript backend does not support transparency; partially transparent artists will be rendered opaque.
In [79]:
df.to_excel('df_after_final_plotting_all_clones.xlsx')
df[df.clone_id_aa.isin(clone_order)].to_excel('df_after_final_plotting_selected_clones.xlsx')
Get leiden distribution for selected plots
In [80]:
#### adjusted stacked barplot for frequency plotting:
def get_color(adata, obs, value):
color = adata.uns[f"{obs}_colors"][
sorted(adata.obs[obs].unique().tolist()).index(value)
]
return color
def plot_frequencies(
adata,
groupby,
subgrouping=None,
subgroup_order=None,
normalize=True,
stacked=True,
order=None,
kind='bar',
bbox_to_anchor=(1, 0.5),
celltype_cat='celltype',
celltypes_to_plot=None, # ← now None by default
celltype_order=None, # ← new: explicit stacking order
colors=None, # ← now None by default
return_df=False,
ax=None, # ← now None, not string
figsize=(10, 5),
legend_columns=1
):
"""Plot frequencies of an observable in a stacked bar chart,
with optional subgrouping and full control over category order.
Parameters
----------
adata : AnnData
groupby : str
subgrouping : str or None
subgroup_order : list-like or None
normalize : bool
stacked : bool
order : list-like or None
kind : str
bbox_to_anchor : tuple
celltype_cat : str
celltypes_to_plot : list-like or None
If provided, restrict to these categories.
celltype_order : list-like or None
If provided, plot stacks in this exact bottom→top order.
colors : list-like or None
return_df : bool
ax : matplotlib Axes or None
figsize : tuple
legend_columns : int
Returns
-------
matplotlib.axes.Axes or DataFrame (if return_df=True)
"""
remove_ax = False
# determine subgroup order if not given
if subgroup_order is None and subgrouping is not None:
subgroup_order = adata.obs[subgrouping].unique().tolist()
# split into subgrouped AnnData objects
if subgrouping is not None:
subgroups = [adata[adata.obs[subgrouping] == x].copy()
for x in subgroup_order]
else:
subgroups = [adata.copy()]
subgroup_order = ['all cells']
# set up axes
if ax is None:
fig, ax_list = plt.subplots(
ncols=len(subgroups) + 1, figsize=figsize, sharey=True
)
ax = list(ax_list)
remove_ax = True
else:
if len(subgroups) > 1:
# inset multiple axes into the supplied ax
ax_list = []
n = len(subgroups)
for i in range(n):
ax_list.append(
inset_axes(
ax,
height="100%",
width=f"{int(1/n*100)}%",
bbox_to_anchor=(i*(1/n), 0, 0.8, 1),
bbox_transform=ax.transAxes
)
)
ax.remove()
ax = ax_list
else:
ax = [ax]
final_colors = []
# loop over each subgroup
for data, axes, group in zip(subgroups, ax, subgroup_order):
# decide which cell-types to plot and in what order
if celltype_order is not None:
# keep only those in data, in the order given
celltypes_plot = [
ct for ct in celltype_order
if ct in data.obs[celltype_cat].unique()
]
else:
if celltypes_to_plot:
# intersection, then sort
celltypes_plot = sorted(
set(celltypes_to_plot) &
set(data.obs[celltype_cat].unique())
)
else:
celltypes_plot = sorted(data.obs[celltype_cat].unique())
# fetch colors if not manually provided
if colors is None and f"{celltype_cat}_colors" in data.uns:
final_colors = [
get_color(data, celltype_cat, ct)
for ct in celltypes_plot
]
else:
final_colors = colors
# build the counts DataFrame
samples = data.obs[groupby].unique().tolist()
df = pd.DataFrame()
for samplename in samples:
vc = data[data.obs[groupby] == samplename].obs[celltype_cat].value_counts()
df[samplename] = vc
# normalize if requested
if normalize:
df = df.div(df.sum(axis=0), axis=1)
# enforce sample order if given
if order is not None:
df = df[order]
# actually plot
plot_kwargs = dict(
kind=kind,
stacked=stacked,
title=group,
grid=False,
ax=axes,
width=0.8
)
if final_colors:
plot_kwargs['color'] = final_colors
df.T[celltypes_plot].plot(**plot_kwargs)
axes.legend(loc='center left',
bbox_to_anchor=bbox_to_anchor,
ncol=legend_columns)
# clean up any extra axes/legends
if len(subgroups) > 1:
for a in ax[:-2]:
a.get_legend().remove()
ax[-1].remove()
elif remove_ax:
ax[-1].remove()
if return_df:
return df
else:
return ax if len(ax) > 1 else ax[0]
In [81]:
df_mouse = pd.DataFrame(adata.obs.mouse.unique()).sort_values(by=0)
ordered_m_list = df_mouse[0].tolist()
In [82]:
m_list_comp = []
for clone in clone_order:
mlist = df[df.clone_id_aa==clone].mouse.tolist()
m_list_comp.append(mlist)
# master list
plotting_order = ['0_TE', '2_MP', '1_EE_like', '3_CE', '4_IE_like', '5_na1', '6_na2']
total = sum(len(sub) for sub in m_list_comp)
#get figure
fig, ax = plt.subplots(nrows = total, figsize = (4, 4*total))
ax_count = 0
for i, clone in enumerate (clone_order):
adata_clone = adata[adata.obs.clone_id_aa==clone].copy()
for mouse in m_list_comp[i]:
adata_mouse = adata_clone[adata_clone.obs.mouse==mouse].copy()
present = adata_mouse.obs['leiden'].cat.categories.tolist()
subset_order = [ct for ct in plotting_order if ct in present]
plot_frequencies(adata_mouse, groupby='mouse', celltype_cat='leiden', colors = leiden_dict, ax = ax[ax_count])
ax[ax_count].set_title(mouse)
ax[ax_count].set_axis_off()
ax_count = ax_count + 1
fig.tight_layout()
fig.savefig(save_figure_sc + 'selected_leiden_stacked_sel_clones_all_clusters.eps')
The PostScript backend does not support transparency; partially transparent artists will be rendered opaque.
In [83]:
obs_list = ['clone_id_aa','shared_mouse','clone_id_aa_size_mouse','norm_clone_id_aa_size_mouse_2','norm_clone_id_aa_size_mouse',
'clone_id_aa_rank','TE_ES','MP_ES','MP_over_TE','TE_over_MP','bias']
In [84]:
df = pd.DataFrame(adata.obs[obs_list])
df.to_excel('./IMMUNITY_obs_df.xlsx')
Add outliers¶
In [85]:
df = adata_sub.obs.groupby(['clone_id_aa', 'mouse']).agg(
shared_mouse = ('shared_mouse', 'first'),
clone_id_aa_size_mouse=('clone_id_aa_size_mouse', 'first'),
norm_clone_id_aa_size_mouse=('norm_clone_id_aa_size_mouse_2', 'first'),
clone_id_aa_rank=('clone_id_aa_rank', 'first'),
TE_ES=('TE_ES', 'first'),
MP_ES=('MP_ES', 'first'),
MP_over_TE=('MP_over_TE', 'first'),
TE_over_MP=('TE_over_MP', 'first'),
bias=('bias', 'first'),
).reset_index()
df = df.dropna()
df = df[df.bias!='not_assigned'].reset_index(drop=True).copy()
### select only top 3 clones for faster plotting
clone_order = order[:3].tolist()
In [86]:
df[df.clone_id_aa==clone_order[0]]
Out[86]:
| clone_id_aa | mouse | shared_mouse | clone_id_aa_size_mouse | norm_clone_id_aa_size_mouse | clone_id_aa_rank | TE_ES | MP_ES | MP_over_TE | TE_over_MP | bias | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 6 | 2282 | m4 | 24.0 | 12.0 | 35.210031 | 7.0 | 2.649306 | 1.297619 | 0.489796 | 2.041667 | eff |
| 7 | 2282 | m5 | 24.0 | 38.0 | 160.941176 | 1.0 | 0.296053 | 0.740132 | 2.5 | 0.4 | unbiased |
| 8 | 2282 | m7 | 24.0 | 29.0 | 129.875598 | 3.0 | 2.574713 | 0.789969 | 0.306818 | 3.259259 | eff |
| 9 | 2282 | m20 | 24.0 | 15.0 | 187.200000 | 1.0 | 0.236364 | 1.915789 | 8.105263 | 0.123377 | mem |
| 10 | 2282 | m21 | 24.0 | 19.0 | 66.358209 | 5.0 | 0.431732 | 2.659774 | 6.160714 | 0.162319 | mem |
| 11 | 2282 | m26 | 24.0 | 19.0 | 23.277487 | 10.0 | 0.49038 | 1.730856 | 3.529621 | 0.283317 | mem |
| 12 | 2282 | m38 | 24.0 | 81.0 | 81.000000 | 5.0 | 1.548135 | 1.062386 | 0.686236 | 1.457225 | unbiased |
| 13 | 2282 | m42 | 24.0 | 33.0 | 88.758621 | 4.0 | 1.1642 | 0.554859 | 0.476601 | 2.098191 | eff |
| 14 | 2282 | m51 | 24.0 | 15.0 | 117.983193 | 2.0 | 0.409375 | 0.288889 | 0.705683 | 1.417067 | unbiased |
| 15 | 2282 | m56 | 24.0 | 76.0 | 259.620438 | 1.0 | 0.583228 | 1.381367 | 2.368486 | 0.422211 | mem |
| 16 | 2282 | m60 | 24.0 | 13.0 | 57.126761 | 3.0 | 1.5 | 1.079365 | 0.719577 | 1.389706 | unbiased |
| 17 | 2282 | m65 | 24.0 | 16.0 | 50.938776 | 5.0 | 0.94375 | 1.155612 | 1.22449 | 0.816667 | unbiased |
| 18 | 2282 | m75 | 24.0 | 39.0 | 147.193548 | 2.0 | 1.225943 | 1.247136 | 1.017287 | 0.983007 | unbiased |
In [87]:
from scipy.stats import gaussian_kde
nrows = len(clone_order)
ncols = len(df[df.clone_id_aa==clone_order[0]].mouse.unique().tolist())
#plotting params
x_all = adata.obsm['X_umap'][:, 0]
y_all = adata.obsm['X_umap'][:, 1]
xmin, xmax = x_all.min(), x_all.max()
ymin, ymax = y_all.min(), y_all.max()
fig, ax = plt.subplots(ncols = ncols, nrows = nrows, figsize = (3 * ncols, 3 * nrows), constrained_layout=True)
#row indexer
y = 0
for clone in clone_order:
#subset dataframe for ordering and scores
mouse_list = df[df.clone_id_aa==clone].mouse.unique().tolist()
#filter for mouse and clone
df_subset = df[np.logical_and(df.mouse.isin(mouse_list), df.clone_id_aa==clone)].copy()
#sort dataframe from highest effector score to unbiased to highest memory score
df_subset['sorted_score'] = df_subset.TE_over_MP.tolist()
df_subset.sorted_score[df_subset.bias == 'mem'] = df_subset[df_subset.bias == 'mem'].MP_over_TE * (-1)
df_subset = df_subset.sort_values(by='sorted_score', ascending = False).copy()
#sorted data
sorted_m = df_subset.mouse.tolist()
sorted_bias = df_subset.bias.tolist()
#start plotting
# col indexer
x = 0
for mouse, bias in zip(sorted_m, sorted_bias):
# Plot background distribution of cells in silver
sc.pl.umap(
adata,
color='leiden',
add_outline=True, outline_width=(0.2, 0), outline_color=('black', 'white'),
s=50,
show=False,
ax=ax[y,x],
frameon=False,
palette=['lightgray'],
legend_loc=None
)
# Extract UMAP coordinates for the subset (clone 'x')
subset = adata[np.logical_and(adata.obs.clone_id_aa == clone, adata.obs.mouse == mouse)]
umap_coords = subset.obsm['X_umap']
if bias == 'eff':
c = 'maroon'
elif bias == 'unbiased':
c = 'black'
else:
c = 'navy'
#get clone size in mouse to adjust threshold
clone_size = df_subset[np.logical_and(df_subset.mouse == mouse, df_subset.bias == bias)].clone_id_aa_size_mouse.values[0]
if clone_size < 20:
kde_thresh = 0.4
levels = 4
else:
kde_thresh = 0.2
levels = 4
#plot density
sns.kdeplot(
x=umap_coords[:, 0],
y=umap_coords[:, 1],
ax=ax[y,x],
fill=True,
color=c,
#cmap=cmap,
bw_adjust = 0.8,
alpha= 0.9, # Adjust transparency if needed
levels = levels,
thresh = kde_thresh,
linewidths = 5,
clip_on = False
)
######### add outliers
# 1. Stack points for gaussian_kde
xy = np.vstack([umap_coords[:, 0], umap_coords[:, 1]])
# 2. Fit & evaluate
kde_model = gaussian_kde(xy, bw_method=0.8)
densities = kde_model(xy)
# 3. Compute the absolute density cutoff
# Seaborn’s `thresh` is relative to the maximum density:
density_cutoff = kde_thresh * densities.max()
# 4. Mask and scatter the “outliers”
outliers = densities < density_cutoff
ax[y, x].scatter(
umap_coords[outliers, 0],
umap_coords[outliers, 1],
s=20, # dot size
color=c, # same color as the fill
edgecolor='white', # white halo for visibility
linewidth=0.5,
alpha=0.8,
label='outlier'
)
#adjust colum for new donor and set title
ax[y,x].set_title('M' + mouse[1:], fontsize = 32, loc='center', y=1.0, pad=-0);
x = x+1
#adjust row for new clone
y = y+1
for ax_ in fig.axes:
ax_.set_xlim(-6, 14)
ax_.set_ylim(0, 18)
fig.canvas.draw()
fig.tight_layout(pad=0.4, w_pad=0.5, h_pad=1.0)
for i in range(nrows):
# Get the bounding box of the leftmost axis in this row (in figure coordinates)
bbox = ax[i, 0].get_position()
# Calculate the vertical center of this axis
y_center = (bbox.y0 + bbox.y1) / 2
# Place text slightly to the left of the axis; adjust the x-offset as needed
fig.text(bbox.x0 - 0.02, y_center, clone_order[i],
va='center', ha='right', fontsize=32, rotation='vertical')
fig.savefig(save_figure_sc + 'selected_kdp_umaps_sel_clones_all_clusters_w_outliers.eps')
WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color. WARNING: Length of palette colors is smaller than the number of categories (palette length: 1, categories length: 7. Some categories will have the same color.
The PostScript backend does not support transparency; partially transparent artists will be rendered opaque.
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